ESI–MS analysis of Cu(I) binding to apo and Zn7 human metallothionein 1A, 2, and 3 identifies the formation of a similar series of metallated species with no individual isoform optimization for Cu(I)

Abstract Metallothioneins (MTs) are cysteine-rich proteins involved in metal homeostasis, heavy metal detoxification, and protection against oxidative stress. Whether the four mammalian MT isoforms exhibit different metal binding properties is not clear. In this paper, the Cu(I) binding properties of the apo MT1A, apo MT2, and apo MT3 are compared and the relative Cu(I) binding affinities are reported. In all three isoforms, Cu4, Cu6, and Cu10 species form cooperatively, and MT1A and MT2 also form a Cu13 species. The Cu(I) binding properties of Zn7-MT1A, Zn7-MT2, and Zn7-MT3 are compared systematically using isotopically pure 63Cu(I) and 68Zn(II). The species formed in each MT isoform were detected through electrospray ionization–mass spectrometry and further characterized using room temperature phosphorescence spectroscopy. The mixed metal Cu, Zn species forming in MT1A, MT2, and MT3 have similar stoichiometries and their emission spectral properties indicate that analogous clusters form in the three isoforms. Three parallel metallation pathways have been proposed through analysis of the detailed Cu, Zn speciation in MT1A, MT2, and MT3. Pathway ① results in Cu5Zn5-MT and Cu9Zn3-MT. Pathway ② involves Cu6Zn4-MT and Cu10Zn2-MT. Pathway ③ includes Cu8Zn4-MT. Speciation analysis indicates that Pathway ② is the preferred pathway for MT2. This is also evident in the phosphorescence spectra with the 750 nm emission from Cu6Zn4-MT being most prominent in MT2. We see no evidence for different MT isoforms being optimized or exhibiting preferences for certain metals. We discuss the probable stoichiometry for MTs in vivo based on the in vitro determined binding constants.


Introduction
Metallothionein (MT) proteins are cysteine-rich proteins involved in Cu(I) and Zn(II) homeostasis, heavy metal detoxification, and protection against oxidative stress.The importance of MTs is emphasized by their presence across multiple domains of life.In mammals, there are four MT isoforms, MT1, MT2, MT3, and MT4 (Fig. 1 ).All mammalian MTs contain 20 cysteines.These cysteines are divided into a β domain containing 9 cysteines and an α domain containing 11 cysteines based on the two domain structures that form when seven divalent metals bind to mammalian MTs. 1 The multiple MT isoforms originate from gene duplication events prior to the evolution of mammals.The first gene duplication event resulted in the divergence of MT4 from the ancestor of MT1, MT2, and MT3.A second duplication event then separated the ancestor of MT1 and MT2 from MT3.Interestingly, while there is one copy of the MT2, MT3, and MT4 genes, there are 13 copies of the MT1 gene in the human genome, where each copy is considered a sub isoform of MT1.Five of these duplicate copies are pseudogenes meaning that they do not encode a functional protein resulting in eight functional sub isoforms of MT1. 2 The Fig. 1 Comparison of human MT1A (Uniprot # P04731 ), MT2 (Uniprot # P02795 ), MT3 (Uniprot # P25713 ), and MT4 (sequence from Moleirinho et al. 2 ) amino acid sequences.Amino acids labeled in red are different compared to the corresponding amino acid in MT1A.Amino acids labeled in blue are similar amino acids to the corresponding amino acid in MT1A.amino acid sequence of MT3 differs significantly compared to the other MT isoforms.It has a threonine insert at position 5 and a hexapeptide insert in the α domain.The CPCP motif (residues 6-9), specific to MT3, results in an additional growth inhibitory function in neurons. 3MT4 also has an additional glutamic acid in the β domain compared to MT1 and MT2.MT4 is suggested to play a role in zinc metabolism during differentiation of stratified epithelial cells. 4n humans, MT1 and MT2 are expressed throughout the body. 5MT2 exhibits higher basal expression than the MT1 sub isoforms, which have different expression patterns depending on the sub isoform. 5MT3 is primarily found in the central nervous system, 6 but has been found to be expressed in other locations as well. 7 , 8T4 expression seems to be the most limited where it is expressed strictly in epithelial cells. 4he presence of multiple MT isoforms and their different expression patterns leads directly to the question of whether these isoforms have different functions and/or metal binding properties.Under normal physiological conditions, MTs tend to be isolated with Zn(II) and Cu(I). 6 , 9 -15Upon exposure to Cd(II), MTs in the liver and kidneys can accumulate Cd(II). 11 , 16 , 17Early research on MT1 and MT2 found that MT1 and MT2 expression responded differently to glucocorticoids, Zn(II), as well as Cd(II).While MT2 was found to be highly inducible by glucocorticoids and Zn(II), MT1A was found to only be highly stimulated by the presence of Cd(II).This led to the hypothesis that MT1 and MT2 have different functions with MT1 serving a protective role and MT2 being involved in the homeostasis of Cu(I) and Zn(II). 18These potentially different functions may arise from differences in the expression or, as some have suggested, differences in the metal bind-ing properties of the two MT isoforms. 19MT2 is less susceptible to proteolytic breakdown compared to MT1. 20 This may also be due to differences in the metallation properties, as metal-thiolate clusters in MT can impede proteolysis. 21MT2 has been shown to bind Zn(II) and Cd(II) ions with higher affinity compared to MT3. 22 Differences in the metal binding character have been reported for mouse MT1, MT2, and MT3 and these isoforms have been described as having either "Cu-thionein character" or "Zn-thionein character." 19 , 23In these studies, the metal binding properties were analysed by expressing the recombinant MT proteins in Escherichia coli ( E. coli ) in the presence of either Zn(II), Cd(II), or Cu(I) and the resulting products were isolated and purified before electrospray ionization (ESI)-mass spectral analysis. 19 , 23While there were no issues isolating Cd 7 -mouse MT2 and Zn 7 -mouse MT2 from the E. coli , the researchers had difficulty isolating Cu-mouse MT2 and concluded that mouse MT2 had a "poor performance for Cu(I) coordination" unlike mouse MT1. 19Our recent results, however, show no issues binding Cu(I) to Zn 7 human MT2 24 or rabbit liver MT2 25 in vitro (and in this paper we demonstrate that there are no issues with Cu(I) binding to apo MT2).It is possible that the poor Cu(I) binding reported previously 19 was due to difficulties in isolating the very air sensitive products.On the other hand, mouse MT3 was reported to be more suited to Cu(I) binding than MT1 and MT2 by the same method. 23However, it is difficult to draw conclusions about the metal binding properties from the methods used due to additional possible complications from variations in metal concentrations within the cells as well as the possibility for disruption of the metallation status upon purification, e.g. through protein oxidation.While in vitro Cu(I) replacement studies of Zn 7 -MTs were carried out, 19 , 23 the authors used pH changes to distinguish Cu(I) and Zn(II) binding in the ESI-mass spectra, which would also lead to metal rearrangement as the structures adopted by Cu(I) have been shown to be very pH dependent. 19 , 23 , 26e have recently reported on the exact Cu, Zn ratios that result upon addition of 63 Cu(I) to 68 Zn 7 -MT1A, 27 68 Zn 7 -MT2, 24 and 68 Zn 7 -MT3 28 and so in our present report we are able to, for the first time, compare the metallation properties of the three isoforms in much greater detail.We note that more extensive summaries of the previous studies of Cu(I) binding to MT1A, MT2, and MT3 were included in the three original publications 24 , 27 , 28 and that a detailed historical record is not repeated here although we do include a commentary on the issues inherent in some of the previous reports with respect to determining exact Cu: Zn ratios bound.
In this paper, we present a comprehensive comparison of the Cu, Zn binding properties of MT1A, MT2, and MT3 through analysis of the species formed after the addition of 63 Cu(I) to the 68 Zn 7 -MT1A, 68 Zn 7 -MT2, and 68 Zn 7 -MT3 as well as the addition of naturally abundant Cu(I) to apo MT1A, MT2, and MT3.We have included novel Cu(I) binding data to apo MT2 to complete the full set of Cu(I) binding data for each of the three major human isoforms.From the array of detailed speciation data for these three prominent human MT isoforms, we can identify recurring species that form starting from the novel Cu 1 Zn 7 -MT1A/2/3.Analysis of the subsequent, cooperatively formed species leads to the proposal that the same two or three metallation pathways are active in each isoform.

Recombinant protein expression and purification
Cd-saturated MT1A, MT2, and MT3 were produced recombinantly using E. coli and purified according to the procedures published Paper | 3 in Melenbacher et al. 27 and Melenbacher and Stillman, 24 , 28 respectively.

MT metallation
All ammonium formate solutions were saturated with argon and contained 0.1 mM tris(2-carboxyethyl)phosphine (TCEP) (Soltec Ventures, Beverly, MA, USA) to prevent oxidation of the thiols.The cadmium was removed from the MT proteins to form the apo MT using a PD10 column (Cytiva) equilibrated to pH 2 using 10 mM pH 2 ammonium formate.The released Cd(II) was separated from the MT through buffer exchange carried out by centrifuging the protein at 4000 ×g using a 3 kDa centrifugal filter (Amicon, Burlington, MA, USA).The pH was increased using dilute argon-saturated NH 4 OH and a second PD10 column was used to bring the pH to neutral pH.While this method completely removes the original cadmium, slight zinc contamination can be observed by the very sensitive electrospray ionization-mass spectrometry (ESI-MS).This is due to the MT scavenging the unavoidable trace contamination of zinc despite acid washing of glassware.In between each step, the protein was also thoroughly evacuated and argon-saturated to avoid oxidation.The concentration of the apo protein was measured by metallating a fraction of the protein sample with cadmium and measuring the absorbance at 250 nm (S-Cd ligand to metal charge transfer band) using a molar absorption coefficient of 89 000 M −1 cm −1 .
68 ZnO was purchased from Trace Sciences International (Richmond Hill, Ontario, Canada) and dissolved in dilute acetic acid (Caledon Laboratory Chemicals, Georgetown, Ontario, Canada) at approximately 60°C.Dilute NH 4 OH was used to increase the pH to 4.1 and the 68 Zn(II) was diluted to a final concentration of 10 mM using Milli Q water.A total of 7.5 mol.eq. of argon-saturated 68 Zn(II) was added to the apo MT to ensure all of the protein was saturated with 68 Zn(II) to form 68 Zn 7 -MT (confirmed by ESI-MS).
The pH of the protein was then adjusted to neutral pH.Cu(I) titrations were carried out using either the apo MT or the 68 Zn 7 -MT (formed as described earlier).When using apo MT, Cu(I) was added as tetrakis(acetonitrile)copper(I) hexafluorophosphate (Millipore-Sigma) in a 10 mM solution in 50% acetonitrile.When adding Cu(I) to the 68 Zn 7 -MT, isotopically pure 63 Cu(I) was used as this allows for the distinction between Cu(I) and Zn(II) by ESI-MS. 27Isotopically pure 63 CuCl 2 was purchased from Trace Sciences International.Glutathione (GSH) (Millipore Sigma) was used to reduce the copper from the 2 + oxidation state to the 1 + oxidation state as reported by Ferreira et al . 29GSH was dissolved in argon-saturated pH 7.4 10 mM ammonium formate and Cu(II) was added resulting in a 3:1 GSH: Cu(II) ratio.The mol. eq. of the reduced 10 mM Cu(I) solution (bound to GSH) were added to the 68 Zn 7 -MT.The ESI-mass spectrum and emission spectrum of the result species were measured after a 10 min equilibration period.For each addition of 63 Cu(I) mentioned, we mean the addition of Cu(I) bound to an unknown number of GSH and the oxidized glutathione (GSSH) product.

ESI-MS methods
Samples were measured by direct infusion into the Bruker Mi-crOTOF II (Bruker Daltonics) in positive ion mode.The parameters used are shown in Table 1 .All samples were rigorously deaerated and argon-saturated to prevent oxidation.The abundance of each species formed from the addition of Cu(I) to the protein was divided by the total recovered protein detected by the ESI-MS and multiplied by 100 to obtain a percentage abundance for each species.The percentage abundance of each species was plotted as a function of the Cu(I) bound to the protein.The 63 Cu, 68 Zn-MT species distributions were determined by simulating the mass spectra of each possible species.The program mMass (version 5.5.0) 30 was used to simulate the isotopic distribution of the apo protein.One or two protons were removed from the apo MT formula for each Cu(I) or Zn(II) bound to the protein, respectively, to mimic the deconvolution calculation where the charge of the metal ion is compensated for by proton loss.The mass of the appropriate number of 68 Cu(I) and 68 Zn(II) ions were added to the apo MT distribution.The mass spectra of species with the same total number of metals but differing Cu: Zn ratios were added in different fractions to identify the composition of the experimentally determined peak.Similar to the method used with the apo protein, the percentage abundance of each Cu, Zn-MT species was plotted as a function of the mol.eq.Cu(I) bound to the protein.

Emission spectroscopy
The emission spectra were measured for the species forming in each step of the 63 Cu(I) titration of Zn 7 -MT1A/2/3 using the Photon Technology International Quanta Master 4 scanning spectrophotometer (Photon Technology International, London, Ontario, Canada).Mass spectra were measured for the same solutions at each point in the titration to identify the species giving rise to the emission spectra.The MT solutions were kept in sealed quartz cuvettes with a septum to avoid oxidation of the protein and deactivation of the triplet excited state.The emission was stimulated by exciting the protein at 280 nm using a Xenon Flash lamp (flashing at 100 Hz) and the emission spectrum was measured from 500 to 900 nm.A yellow filter was used to eliminate scatter from the exciting beam.The emission slits were set to 10 nm and the exciting slits were set to 20 nm.The emission intensities were calibrated for the sensitivity of the GaAs phototube by measuring an HL-2000 HP Light Source (Ocean Insight, Orlando, FL, USA).The emission spectra at certain Cu(I) mol.eq. were compared for MT1A, MT2, and MT3.

Binding constant simulations
The program Hyperquad Simulation & Speciation (HySS) was used to simulate a series of relative pH dependent, apparent cumulative binding constants, β, for the species forming upon the titration of apo MT1A, apo MT2, and apo MT3.The apparent β values were adjusted until the calculated speciation matched the experimental speciation from the ESI-mass spectral data.The apparent β values for undetected intermediate species were set at the highest values possible where they do not appear in the simulation.The pH dependent, apparent stepwise log K F binding constants for species with n Cu(I) ions were calculated from the apparent log β values (log β n -log β n -1 ).Since HySS cannot handle the large β values that would result from the affinities expected for MT, only relative log β values are simulated.The apparent K F values were then scaled so that the K F for Cu 6 -MT was set to the value of Cu 6 -MT determined by Scheller et al .(also at pH 7.4). 26o demonstrate the unavoidable uncertainty when intermediate species are undetectable due to the cooperativity of certain species, simulations are shown that demonstrate the effect of changing the K F of the unobserved species Cu 9 -MT2 vs. an observed species, Cu 10 -MT2.

Cu(I) metallation of apo MT1A, apo MT2, and apo MT3
Before comparing the more complicated speciation that arises from the addition of 63 Cu(I) to 68 Zn 7 -MT1A/2/3, we first compare the speciation that results from the addition of Cu(I) to apo MT1A, 31 apo MT2, and apo MT3, 28 which may occur in the body with newly synthesized MT.The ESI-mass spectral data for the titration of Cu(I) into apo MT2 are shown in Fig. 2 .The titration closely resembles the Cu(I) titration of MT1A 26 , 31 ; however, the Cu 15 -MT2 species is more prominent than in MT1A.Of note, Cu(I) titrations of rabbit liver MT revealed strong features in the circular dichroism (CD) spectra identified for a Cu 15 species. 32Russell et al .reported similar species by ESI-MS after the addition of Cu(II).The authors reported that the use of the reducing agent, TCEP, would reduce the Cu(II) to Cu(I). 33We have not found the presence of TCEP to be sufficient in reducing Cu(II) to Cu(I) and so it is possible that some of the MT may have oxidized in that previous study.Conducting the Cu(I) titration of MT2 under the exact same conditions as we have reported for MT1A 31 and MT3 28 allows for a better comparison of the metallation properties.
Figure 3 shows the abundance of each species forming during the titration of apo MT1A (Fig. 3 A), apo MT2 (Fig. 3 B) and apo MT3 (Fig. 3 C) detected by ESI-MS as a function of the mol.eq. of Cu(I) bound to the protein.The speciation data were simulated using the software HySS.The titrations results for all three isoforms are similar with the formation of Cu 4 -MT1A/2/3, Cu 6 -MT1A/2/3, and Cu 10 -MT1A/2/3 in each protein at the approximately same Cu(I) loading.
The Cu 4 -MT1A/2/3 and Cu 6 -MT1A/2/3 species are proposed to have Cu 4 and Cu 6 clusters in the β domain.The Cu 10 -MT1A/2/3 species is a combination of a Cu 6 cluster in the β domain and a Cu 4 cluster in the α domain. 27 , 31he Cu(I) titration of apo MT3 differs significantly from the data for apo MT1A and apo MT2 in several respects.First, the relative abundance of the Cu 4 -species compared with the cooperatively formed Cu 6 -species for the MT3 is significantly less than observed for MT1 and MT2.Second, in MT1A and MT2, Cu 13 -MT1A/2 forms cooperatively after Cu 10 -MT1A/2, whereas in MT3, a series of species with increasing ratios of Cu(I) form non-cooperatively in low abundance with no species being preferred over the others.The Cu 13 -MT1A/2 species are proposed to be formed from a combination of a Cu 6 cluster in the β domain and a Cu 7 cluster in the α domain.The absence of a prominent Cu 13 -MT3 species suggests that the Cu 7 cluster cannot form in the α domain of MT3, possibly due to the presence of the acidic loop, a hexapeptide insert of acidic amino acids (Fig. 1 , residues 55-60).This insertion may destabilize the Cu 7 -α cluster.While it is possible that the Cu 13 -MT3 species specifically does not ionize as well as the Cu 13 -MT1A and Cu 13 -MT2 species or the prior Cu 10 -MT3 species, we have not found evidence for noticeable changes in ionization efficiencies when carrying out stepwise metallation of the same protein.In the past we have used emission spectroscopy, 31 ultraviolet-absorption spectroscopy, 34 , 35 and CD spectra 24 as controls to confirm that the species ratios determined by the mass spectrometry fit the solution spectroscopic data.
HySS was used to simulate relative pH dependent, apparent cumulative binding constants, log β, using a series of cumulative reactions (Table 2 ).The pH dependent, apparent K F values for species with n Cu(I) ions for the stepwise reactions corresponding to the cumulative reactions shown in Table 2 were calculated by subtracting β n -β n −1 .In the subsequent text and figures, all log β and log K F values are the apparent constants determined at pH 7.4.We note that due to constraints with the program, the absolute values cannot be simulated and instead the resulting K F  28 Log K F values for key species shown in Fig. 6 and Table 3 .
values are scaled to literature values.The values of these log β were adjusted until the simulated speciation fit the experimental speciation (Fig. 3 ).It is chemically reasonable to predict that each metal binds one by one, though due to the cooperative properties of subsequent species, not all of the intermediate species form in detectable concentrations (the effect of a cooperative K F of a species is to deplete the concentration of any species prior to the cooperative one).For all three isoforms, the β values for these intermediate species (i.e.Cu 1-3 -MT) which do not form in detectable amounts were set to the highest possible value before the species was evident in the simulation.Figure 4 demonstrates how log K F of these unobserved species can be modified without significant impact on the overall speciation leading to much higher uncertainty in the log K F values for these unobserved, but necessary, intermediates than for the observed species.With log K 9 set between 4.93 (Fig. 4 , solid line) and 14.09 (Fig. 4 , dashed line), no detectable amounts of Cu 9 -MT2 form, as with the experimental data (Fig. 3 B).Changing log K 9 within this range has no impact of the species that form before and after, Cu 8 -MT2 and Cu 10 -MT2, respectively, as seen by the two sets of data lying on top of each other in Fig. 4 .The effect of changing log K 9 while leaving the remaining K F values the same is variation in log K 10 as log K 10 is calculated by subtracting log β 9 from log β 10 .While we demonstrate this variability in K 9 , we do however note that a log K F of 4.93 is not reasonable for Cu(I) binding to thiols and we expect that the value is closer to the higher end of the spectrum.A log K 10 of 19.91 agrees more readily with the published value for Cu 10 -MT, which was determined at pH 7.4 as well. 26hereas, adjusting the log K F of one observed species, e.g.Cu 10 , by even just ±1 without changing any other of the other log K F values, significantly impacts the speciation simulation, which results in different mass spectral profiles (Fig. 5 ). Figure 5 A shows the speciation where log K 10 = 19.91,which is the value that fits the experimental data shown in Fig. 3 B. The speciation shows that Cu 6 -MT2, Cu 10 -MT2, and Cu 13 -MT2 all reach a similar abundance throughout the titration.Figure 5 B shows the mass spectral profile for this speciation with 9.7 mol.eq.Cu(I).The most abundant species in the spectrum is Cu 10 -MT2.Decreasing log K 10 by 1 to 18.91 (log K 10 ʹ) results in significant changes to the speciation with Cu 10 -MT2 no longer reaching approximately the same abundance as Cu 6 -MT2 and Cu 13 -MT2 throughout the course of the titration.Now, Cu 10 -MT2 only reaches a maximum abundance of about 35% (Fig. 5 C). Figure 5 D shows the simulated mass spectral profile for this speciation with 9.7 mol.eq.Cu(I).Compared to Fig. 5 D, if log K 10 is decreased, then more Cu 6 -MT2 and Cu 11-13 -MT2 would be found in the mass spectrum.Figure 5 E shows the resulting speciation if log K 10 is increased by 1 to 20.91 (log K 10 ʺ).This results in the formation of much more Cu 10 -MT2 and much less Cu 6 -MT2 and, to a lesser extent, less Cu 13 -MT2 throughout the titration.Figure 5 F shows how the mass spectral profile of this speciation with 9.7 mol.eq.Cu(I) has predominantly Cu 10 -MT2.Overall Figs. 4 and 5 show how the abundance of the observed species are much more sensitive to changes in the log K F values than the unobserved intermediates that are assumed to form in the metallation pathway.This results in significantly greater errors in the log K F values of those unobserved species in our model where the Cu(I) ions bind sequentially.
The HySS fits previously reported for MT1A 31 and MT3 28 were refit in the same manner so that the K F values could be compared (Fig. 3 ).Based on the similarities in the formation of Cu 6 -MT in the speciation for all three isoforms at pH 7.4, the log K F values were scaled so that the log K F for Cu 6 was 20.38, the value determined for MT1A by Scheller et al . 26at pH 7.4 (Fig. 6 ).
The general trend of the log K F values is relatively similar for all three isoforms.There are slight differences in the log K F for equivalent species forming in the three isoforms, which may be due to the error discussed earlier.The cooperativity of the Cu 4 -MT, Cu 6 -MT, and Cu 10 -MT is evident by the fact that the intermediates between these clustered species are not observed to form   3 .
in the ESI-mass spectral data.Due to this, we have chosen not to show the log K F values for these unobserved species in Fig. 6 as the error on the values is much higher than for the observed species as demonstrated earlier.To keep the analysis systematic between isoforms, the log β values used for the experimentally undetected species are the largest possible values that do not lead to detectable formation of that species.This may underestimate the values of log K 4 , K 6 , and K 10 .We report the log K F values for the observed species up to Cu 15 -MT for MT1A, MT2, and MT3 in Table 3 .We have added positive error bars on Fig. 6  In all three isoforms, the K F values for Cu 6 -MT are higher than those for Cu 4 -MT and Cu 10 -MT, which explains the prominence of Cu 6 -MT early in the titration over Cu 4 -MT and Cu 10 -MT.Because the K F for Cu 10 -MT is lower than that for Cu 6 -MT, there is only sig-nificant formation of Cu 10 -MT once all of the apo protein has been used to form Cu 6 -MT.In MT1A and MT2, the Cu 13 -MT1A/2 species forms with very high abundance compared to Cu 11 -MT1A/2, Cu 12 -MT1A/2, Cu 14 -MT1A/2 and Cu 15 -MT1A/2 (Fig. 3 A, B).This is due to the K F values increasing from Cu 11 up to Cu 13 -MT1A/2 before dropping with Cu 14/15 -MT.In MT3, the K F for Cu 12 -MT3 is higher than Cu 11 -MT3, Cu 13 -MT3, Cu 14 -MT3, and Cu 15 -MT3.However, the Cu 12 -MT3 species does not accumulate in MT3 to the same extent as Cu 13 -MT1A/2 due to the K F values for Cu 13 -MT3, Cu 14 -MT3, and Cu 15 -MT3 being much more similar in magnitude compared to the difference between the K F for Cu 13 -MT1A/2 and the K F for Cu 14 -MT1A/2.

Cu(I) metallation of each Zn 7 -MT isoform forms the Cu 1 Zn 7 -MT species first
The ESI-mass spectra reveal that the first species to form in MT1A, MT2, and MT3 is Cu 1 Zn 7 -MT (Fig. 8 ).This was unexpected as it was thought that the MT was saturated with seven divalent metals.Evidently, Cu(I) can bind to Zn 7 -MT without displacement of the Zn(II) which implies that the two domain structure set up by the Zn(II) has an available binding site for the Cu(I) ion in all three isoforms.It has not been determined where the Cu(I) ion is binding.It is possible that there are multiple structures for the Cu 1 Zn 7 -MT species.This significant result has implications in the formation of mixed metal species when low levels of Cu(I) exist with high cellular levels of Zn(II), because these data show that the Cu(I) can bind to the saturated Zn-MT.
It is only through the use of native ESI-MS methods, where all of the species in a solution can be disentangled, that the Cu 1 Zn 7 -MT species can be easily detected, unlike with other techniques that can only provide average stoichiometries.We note, as commented earlier, that we have no evidence for different metallated species of MT having different ionization efficiencies.Therefore, we expect the relative abundances of species detected in the ESImass spectra to align with the solution concentrations in these Cu(I) metallation studies.Figure 9 shows the correlation between the formation of the Cu 1 Zn 7 -MT and the loss of Zn 7 -MT in MT1A, MT2, and MT3.These data show that the fractions of the Cu 1 Zn 7 -MT formed are greatest for MT3, followed by MT1A, and then MT2.Palumaa et al .have reported that Cd 8 -MT3 and Zn 8 -MT3 form more readily compared the corresponding species in MT2. 22We have also observed the formation of Zn 8 -MT3 but not Zn 8 -MT1A Fig. 8 Speciation resulting from 63 Cu(I) addition to 68 Zn 7 -MT1A (A), 68 Zn 7 -MT2 (B), and 68 Zn 7 -MT3 (C) as determined from the corresponding ESI-mass m/z data (reported in Melenbacher et al. 27 and Melenbacher and Stillman 24 , 28 ).Only key species are shown for clarity.Full speciation shown in Supplementary Fig. S1.Abundances calculated from ESI-MS data as first reported in Melenbacher et al. 27 and Melenbacher and Stillman. 24 , 28 Zn 8 -MT2 (Fig. 9 ).We note that slight excess Zn(II) was added to apo MT to ensure that all of the protein became metallated with seven Zn(II) ions.With MT3, this resulted in the formation of a small amount of Zn8-MT3.The acidic loop in MT3 may allow for an eighth metal to bind more easily.Significantly, the stoichiometries for the dominant species forming in the next metallation steps after Cu 1 Zn 7 -MT in MT1A, MT2, and MT3 are essentially the same for the three isoforms with Cu 5 Zn 5 -MT1A/2/3, Cu 6 Zn 4 -MT1A/2/3, Cu 9 Zn 3 -MT1A/2/3, and Cu 10 Zn 2 -MT1A/2/3, all being prominent species (Fig. 8 ).An analysis of the relative binding constants for these species forming in MT2 revealed that these species form cooperatively.This is evident by the increase in the relative binding constants for these species compared to the previous species. 24Because of the similar speciation profiles, it is clear that the same is expected for the Cu(I) metallation of Zn 7 -MT1A and Zn 7 -MT3.As Cu(I) binds to the protein, the number of available binding sites decreases.This would result in a decrease in the relative binding constants due to the decreasing statistical likelihood of binding if the binding was non-cooperative or random. 31The speciation of non-cooperative binding is characterized by a binomial distribution of species centered on the average mol.eq. of the metal ion added, as evident by the completely non-cooperative binding of six As(III) ions to apo MT1A 35 and apo MT3. 34W ith regards to MTs, we understand cooperativity as being the formation of clustered species, e.g.Zn 3 S 9 and Zn 4 S 11 36 and in this paper Cu 6 S 9 ( β)Zn 4 S 11 ( α).

The key species forming after
While the Cu, Zn-MT species that form are similar for MT1A, 27 MT2, 24 and MT3, 28 there are differences in the relative abundances of the species resulting in different overall metallation profiles.The main difference is the prominence of the Cu 1 Zn 7 -MT, Cu 6 Zn 4 -MT, and Cu 10 Zn 2 -MT species.

Analysis of MT1A, MT2, and MT3 speciation through ESI-MS led to the determination of multiple separate pathways for stepwise Cu(I) binding
The coexistence of two pairs of species with the same total number of metals bound (10 metals in Cu 5 Zn 5 -MT2 and Cu 6 Zn 4 -MT2 and 12 metals in Cu 9 Zn 3 -MT2 and Cu 10 Zn 2 -MT2) led us to propose two parallel pathways in our detailed analysis of the speciation for Cu, Zn-MT2.Cu(I) metallation pathway 1 forms Cu 5 Zn 5 -MT2 and Cu 9 Zn 3 -MT2 and pathway 2 forms Cu 6 Zn 4 -MT2 and Cu 10 Zn 2 -MT2. 24The presence of this same series of species in MT1A and MT3 allow us to now propose that Cu(I) metallation follows similar pathways in these isoforms as well (Scheme 1 ).The bolded species are the major species of each pathway and the non-bolded species are proposed intermediates.Comparison of the speciation for MT1A, MT2, and MT3 reveals that certain species form with remarkably similar abundances over the same range of Cu(I) bound (Fig. 10 ).By focusing on one species at a time, multiple pathways can be suggested for each of MT1A, MT2, and MT3.We note that the identification of multiple pathways is only possible with the use of isotopically pure 63 Cu(I) and 68 Zn(II) as this allows us to distinguish species from separate pathways with the same total number of metals, but differing Cu: Zn ratios (ex.Cu 5 Zn 5 -MT and Cu 6 Zn 4 -MT).

The significant role of Cu 1 Zn 7 -MT
We propose that after the formation of Cu 1 Zn 7 -MT, subsequent Cu(I) metallation can follow one of several pathways.The fact that this species branches off into multiple pathways might mean that there are multiple structures for the initial Cu 1 Zn 7 -MT species.
Pathway 1 The first species to form from Cu 1 Zn 7 -MT in pathway 1 is Cu 3 Zn 6 -MT (Fig. 10 A).The exchange of one Zn(II) ion for two Cu(I) ions conserves the charge of the molecule.It was seen in MT1A that the species forming all contain a cationic charge from the metal within a certain range. 27The replacement of one Zn(II) ion by two Cu(I) ions is seen again with the formation of Cu 5 Zn 5 -MT (Fig. 10 B) from Cu 3 Zn 6 -MT.The distribution and abundance of the Cu 5 Zn 5 -MT remains constant with all three isoforms.Next, we propose that one Cu(I) ion binds to Cu 5 Zn 5 -MT to form Cu 6 Zn 5 -MT (Fig. 10 C) and then two Cu(I) ions replace two Zn(II) ions to form Cu 8 Zn 3 -MT (Fig. 10 D).The next major product to form in pathway and MT3.This indicates that the relative binding constants (K F ) for the species in pathway 1 are approximately the same for these structures across all three MT isoforms.
Pathway 2 While the abundances of the species forming in pathway 1 remain relatively constant across MT1A, MT2, and MT3, the species forming in pathway 2 show isoform-dependent differences indicating different relative K F values for different human MT isoforms.We show in Fig. 5 that the value of a stepwise binding constant, K F , relative to the other species affects the relative abundance of a species.We propose that the first product of pathway 2 is Cu 6 Zn 2 -MT (Fig. 9 ).We propose that the next species to form in pathway 2 is Cu 4 Zn 5 -MT (Fig. 10 F).Equal fractions of Cu 4 Zn 5 -MT1A and Cu 4 Zn 5 -MT2 form with slightly higher fractions of Cu 4 Zn 5 -MT3 forming.
The next species proposed to form from this pathway is Cu 6 Zn 4 -MT (Fig. 10 G).The abundance of this species is highest in MT2, followed by MT1A, and then MT3.This suggests that the K F for Cu 6 Zn 4 -MT formation follows the trend of MT2 > MT1A > MT3.We note here that because the formation of the Cu(I) is through reduction by GSH, there will be excess GSH over the μM concentration of the Cu(I) added.This GSH will compete for the Zn(II) that is released from the MT as a result of Cu(I) binding. 37This competition becomes more significant with higher ratios of Cu(I) bound, but under those conditions, the Cu(I) binding dominates.The experimental evidence from our data is that the GSH does not exert a significant impact because even with 14 Cu(I) ions, one Zn(II) ion remains bound to MT, which we would have expected to be removed by the GSH.
Cu(I) binding to Cu 6 Zn 4 -MT results in Cu 7 Zn 4 -MT (Fig. 10 H).This species forms in similar abundances in MT1A and MT2.Slightly less Cu 7 Zn 4 -MT forms in MT3.This species is consumed to form Cu 9 Zn 2 -MT (Fig. 10 I).The next species to form after Cu 9 Zn 2 -MT is Cu 10 Zn 2 -MT (Fig. 10 J).Approximately equal amounts of Cu 10 Zn 2 -MT forms in MT1A and MT3, indicating that the binding constants for the two isoforms of this species are similar.There is an increase in the amount of Cu 10 Zn 2 -MT2 that forms, which suggests that its K F is slightly higher than for MT1A and MT3.
Pathway 3 We propose that Cu 8 Zn 4 -MT (Fig. 10 K) may be part of a third pathway.Based on data from Zn 7 -MT3 carried out at pH 7.8, the Cu 8 Zn 4 -MT may form from Cu 5 Zn 5 -MT3. 28

Emission spectra indicate similar Cu(I)-cysteine cluster formation in MT1A, MT2, and MT3
We show that the emission bands are dependent on the cluster stoichiometry through parallel ESI-MS and emission experiments on MT1A, MT2, and MT3.Species with the same Cu: Zn stoichiometry have emission spectral bands at similar emission wavelengths, regardless of the MT isoform.Figure 12 shows the ESI-mass spectra indicating the presence of varying ratios of Cu 5 Zn 5 -MT and Cu 6 Zn 4 -MT (left) and the corresponding emission spectra (right) for these species in MT1A (Fig. 12 A), MT2 (Fig. 12 B), and MT3 (Fig. 12 C).The emission at 650-670 nm is attributed to the Cu 5 Zn 5 -MT species and the emission with a band center at approximately 750 nm is from the Cu 6 Zn 4 -MT.The relative intensity of the 750 nm band correlates with the abundance of Cu 6 Zn 4 -MT found in the ESI-mass spectral data.The similarity in the emission wavelengths confirm the very high reliability of the solution-state emission in identifying the presence of specific Cu n S m clusters.The slight shift in the wavelength of the Cu 5 Zn 5 -MT3 cluster compared to the equivalent cluster in MT1A and MT2 may be due to distortions in the cluster caused by the CPCP motif found only in MT3.It is only by combining the room temperature solution phosphorescence measurements with the ESI-mass spectral measurements can the stoichiometries of the clusters giving rise to the emission be determined.The emission spectra are actually more sensitive to the presence of multiple species as the ESI-mass spectra can only be disentangled with the use of both the isotopically pure metals and mass spectral simulations.
Figure 13 shows the emission profiles for the first six Cu(I) ions bound to Zn 7 -MT for MT2 (Fig. 13 A), MT1A (Fig. 13 B), and MT3  27 and Melenbacher and Stillman. 24 , 28ig. 13 C).While the emission wavelengths are similar for MT1A, 27 MT2, 24 and MT3, 28 there are differences within the first six mol.eq.Cu(I) bound that reflect the different ratios of M 10 species forming.All three isoforms show an increase in the emission at 650/670 nm due to the formation of Cu 5 Zn 5 -MT.The formation of Cu 6 Zn 4 -MT can be tracked through the presence of the 750 nm emission.The similarities in the emission wavelengths indicate that the excited state properties of the clusters are relatively similar across MT1A, MT2, and MT3.
MT2 exhibits the greatest intensity of the 750 nm emission, followed by MT1A, and finally MT3.This follows the trends in the ESImass spectral data for the formation of Cu 6 Zn 4 -MT vs. Cu 5 Zn 5 -MT (Fig. 8 ). Figure 13

MT isoforms do not show signs of being optimized for different metals
Analysis of ESI-mass spectral data allows for novel comparisons of the species forming in human MT1A, MT2, and MT3 after the addition of Cu(I) to the apo or Zn 7 forms of the proteins.These conclusions concerning speciation are supported by the room temperature Cu n S m cluster-dependent emission spectra.A series of similar Cu(I)-thiolate clusters form following the stepwise addition of Cu(I) to the apo proteins of all three isoforms.The species forming when 63 Cu(I) is added to 68 Zn 7 -MT1A, 68 Zn 7 -MT2, and 68 Zn 7 -MT3 are more complicated.The first species to form is Cu 1 Zn 7 -MT and then a series of species with the same stoichiometries form following one of three separate pathways for each of the three isoforms.Despite the differences in the non-cysteine amino acids between the three isoforms, our data suggest that it is the conserved positions of the cysteines that largely control the exact speciation.However, there are subtle differences in the abundance of some species.This indicates that there are slight isoform-dependent differences that control the adopted metallation pathway though all three pathways exist in each isoform.Even with these pathway differences, we find no evidence for Cu(I) binding being optimized for one MT isoform over the other when starting with either the apo or Zn 7 form of MT1A, MT2, and MT3.While the data show a lack of Cu 13 -MT when Cu(I) binds to apo MT3 unlike MT1A and MT2, we do not expect this difference to be significant in vivo where additional metals would induce the synthesis of additional MT, effectively decreasing the metal: MT ratio.
Our conclusions differ significantly from those reported for mouse MT1, MT2, and MT3. 19 , 23While our systematic comparison of Cu(I) binding to Zn 7 -MT1A, Zn 7 -MT2, and Zn 7 -MT3 examines the binding properties and species formation in vitro , the methodology used in the mouse MT studies involved synthesizing recombinant MT in the presence of excess Cd(II), Zn(II), or Cu(I) in the E. coli overexpression system before isolating and purifying the protein from that cellular milieu.Those authors concluded that mouse MT2 was not optimized for Cu(I) coordination after experiencing difficulty isolating Cu(I) containing MT2.As a result, Atrian et al .labeled mouse MT2 as a Zn-thionein. 19We would expect that if MT2 was truly less optimized for Cu(I) than the other isoforms that we would see different species forming when Cu(I) was added to the apo or Zn 7 forms of the protein compared to MT1A and MT3 and so we conclude that MT2 is no less optimized for binding Cu(I) than MT1A or MT3.The binding of Cu(I) to Zn 7 -MT2 is actually more cooperative than for Zn 7 -MT1A and Zn 7 -MT3 as seen by the preferential formation of Cu 6 Zn 4 -MT2 over Cu 5 Zn 5 -MT2 compared to MT1A and MT3.Our group has also previously published Cu(I) binding studies of rabbit liver MT2 25 , 38 suggesting that this is not just a property of human MT.The differences found in those previous reports by others may be explained by difficulties in purifying Cu-MTs, a very air sensitive form of MT, from the E. coli .Contrasting results to the study by Atrian et al . 19were also found by Sakurai et al . 39This study separated MT isoforms 1 and 2 isolated from the livers of rats using polyacrylamide-coated capillary zone electrophoresis.The study analysed the MT content from livers of Long-Evans cinnamon (LEC) rats, an animal model

Fig. 2
Fig. 2 ESI-mass spectral data for the titration of Cu(I) into 50.1 μMapo MT2 at pH 7.4.The full MT2 protein is observed at 7325 Da in the mass spectrum.The main species forming after the addition of Cu(I) are Cu 4 -MT2, Cu 6 -MT2, Cu 10 -MT2, Cu 13 -MT2, and Cu 15 -MT2.A fraction of the protein has had the first "GSM" amino acids cleaved from the protein and is seen at 6795 Da (denoted Apo*).These amino acids are not part of the native protein sequence and do not affect metal binding.The Cu(I) metallation of this cleaved MT2 protein mirrors that of the full MT2 protein and the main species that form are Cu 4 *, Cu 6 *, Cu 10 *, Cu 13 *, and Cu 15 *.The masses of these species are 531 Da lower than the corresponding species in the full MT2 protein.The metal equivalences noted on the figure refer to the amount of Cu(I) bound to the protein.

Fig. 3
Fig. 3 Speciation resulting from Cu(I) addition to apo MT1A, MT2, and MT3.(A) Experimental speciation (symbols) and simulated HySS speciation (lines) for Cu(I) binding to apo MT1A.Data originally published in Melenbacher et al . 31(B) Experimental speciation (symbols) and simulated HySS speciation (lines) for Cu(I) binding to apo MT2.(C) Experimental speciation (symbols) and simulated HySS speciation (lines) for Cu(I) binding to apo MT3.Data originally published in Melenbacher and Stillman.28Log K F values for key species shown in Fig.6and Table3.

Fig. 4
Fig. 4 Simulated speciation for two values of log K 9 .Solid lines: Overall speciation if log K 9 = 4.93 and log K 10 = 29.31.Dashed lines: Overall speciation if log K 9 = 14.09 and log K 10 = 19.91.Remaining log K F values are as reported in Table3.
for Cu 4 -MT, Cu 6 -MT, and Cu 10 -MT as these values are quite conservative due to the use of the largest possible log β values for Cu 3 -MT, Cu 5 -MT, and Cu 9 -MT.For Cu 11 -Cu 15 -MT, the error comes from the error in the fit, which we have indicated with smaller positive and negative error bars.

Fig. 5
Fig. 5 Simulated speciation and mass spectral profiles for varying log K 10 values.(A) Simulated speciation where log K 10 = 19.91, the value found to best simulate the experimental data in Fig. 3 B. (B) Simulated mass spectral profile for the speciation in A with 9.7 mol.eq.Cu(I).(C) Simulated speciation where log K 10 ʹ = 18.91(i.e.log K 10 − 1).(D) Simulated mass spectral profile for the speciation in C with 9.7 mol.eq.Cu(I).(E) Simulated speciation where log K 10 ʺ = 20.91 (i.e.log K 10 + 1).(F) Simulated mass spectral profile for the speciation in E with 9.7 mol.eq.Cu(I).

Scheme 1
Scheme 1 Proposed pathway of species forming from human Zn 7 -MT for MT1A, MT2, and MT3 adapted from the pathway proposed for MT2 by Melenbacher and Stillman.24

Fig. 13
Fig. 13 Room temperature emission for the addition of 63 Cu(I) to 68 Zn 7 -MT1A/2/3.(A) Emission spectra measured for the addition of 63 Cu(I) to 68 Zn 7 -MT2 at pH 7.4.(B) Emission spectra measured for the addition of 63 Cu(I) to 68 Zn 7 -MT1A at pH 7.4.(C) Emission spectra measured for the addition of 63 Cu(I) to 68 Zn 7 -MT3 at pH 7.8.(D)Normalized emission spectra for MT1A (black), MT2 (red), and MT3 (blue) when 2.9-3.4 mol.eq.Cu(I) is bound to the protein.Data originally reported in Melenbacher et al.,27 and Melenbacher and Stillman.24 , 28 D shows the emission spectra resulting from of 3.4 mol.eq.Cu(I) bound to Zn 7 -MT1A (Fig.13D, black), 3.4 mol.eq.Cu(I) bound to Zn 7 -MT2 (Fig.13 D, red), and 2.9 mol.eq.Cu(I) bound to Zn 7 -MT3 (Fig.13 D, blue).It is clear in the emission that MT2 forms Cu 6 Zn 4 -MT very early in the titration and in greater fractions compared to MT1A and MT3.These emission spectra support the ESI-MS data that show the formation of Cu 6 Zn 4 -MT through pathway 2 is preferred in MT2 more than MT1A and MT3.The emission spectra for the remainder of the titration are very similar to MT1A and MT3.

Table 2 .
Series of cumulative and stepwise reactions used for determining binding constants